Purification and characterization of A61: An angiostatin-like plasminogen fragment produced by plasmin autodigestion in the absence of sulfhydryl donors

Geetha Kassam, Mijung Kwon, Chang Soon Yoon, Kenneth S. Graham, Mary K. Young, Stefan Gluck, David M. Waisman

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000) Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys78-Lys468. To avoid confusion with the plasmin(ogen) fragment, angiostatin® (Lys78-Ala 440), we have named this protein A61. Unlike angiostatin, A61 was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A61 bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A61 was composed of 21% β-strand, β-turn, 18% 31-helix and 8% 310-helix. A61 was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A61 by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A61 was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a non-pathological setting.

Original languageEnglish (US)
Pages (from-to)8924-8933
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number12
DOIs
StatePublished - Mar 23 2001

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this